Physical interactions define how a TCR recognizes an antigen, but not the mechanism. How that interaction results in specific T cell responses is unknown. The primary critical event that determines whether a cytolytic T cell (CTL) kills an antigen-presenting cell is the interaction between the T cell receptor (TCR) and the peptide/MHC complex (pMHC). We have observed that the xenoreactive pair of p1049/A2 and AHIII 12.2 TCR dock in an orthogonal rather than diagonal orientation seen in other TCK:pMHC pairs. We hypothesize that this unorthodox binding is a result of the absence of T cell selection on HLA-A2.1 by the AHIII12.2 T cell. A large panel of peptides that bind to the syngeneic MHC H-2Db and are recognized by AHIII12.2 has been examined. A second set of peptides, recognized by the P14 T cell, has also been tested. These two sets of peptides and two TCR will be used to probe the biochemical parameters that control T cell activity. There are significant disagreements as to whether affinity (KD) or microscopic rate constants (koff or kon) between pMHC and TCR are the key factors that control T cell activity. We hypothesize that the disagreements stem from the small number of observations made to date of pMHC binding constants to individual TCR. This application explores the physical interactions between pMHC and TCR using a well-defined T cell model systems and a large set of pMHC ligands. This approach is powerful because of the large sample size of pMHC to be assayed and because an interdisciplinary set of physical tools and immunological assays will be used. These tools include: protein crystallography, surface plasmon resonance (SPR) and site-directed mutagenesis. The biophysical measurements will be correlated with T cell responses such as: Ca flux, proliferation, cytokine secretion and cytolytic activity. A hypothesis for a quantitative model of T cell responses derived from the physical interactions of TCR and pMHC will be tested. In Aim 1, we ask whether non-diagonal orientations are common to T cells that have not gone through thymic selection on the restricting MHC. In aim 2, which binding properties between pMHC and TCR are found to be predictive of T cell responses is studied. In Aim 3, we extend our observations to autoimmune diabetes and determine if autoimmune dual-reactive TCR interact with pMHC in a diagonal fashion like that seen for A2 binding to AHIII12.2. These studies will prove to be useful in immunological therapy of autoimmunity, chronic viral infection, cancer and transplantation.